Concentration- and Time-Dependent Effects of Benzalkonium Chloride in Human Lung Epithelial Cells: Necrosis, Apoptosis, or Epithelial Mesenchymal Transition

Benzalkonium chloride (BAC), an antimicrobial agent in inhalable medications and household sprays, has been reported to be toxic to pulmonary organs. Although cell membrane damage has been considered as the main cytotoxic mechanism of BAC, its concentration- and time-dependent cellular effects on lung epithelium have not been fully understood. In the present study, human lung epithelial (H358) cells were exposed to 0.2–40 μg/mL of BAC for 30 min or 21 days. Cell membranes were rapidly disrupted by 30 min exposure, but 24 h incubation of BAC (4–40 μg/mL) predominantly caused apoptosis rather than necrosis. BAC (2–4 μg/mL) induced mitochondrial depolarization, which may be associated with increased expression of pro-apoptotic proteins (caspase-3, PARP, Bax, p53, and p21), and decreased levels of the anti-apoptotic protein Bcl-2. The protein expression levels of IRE1α, BiP, CHOP, and p-JNK were also elevated by BAC (2–4 μg/mL) suggesting the possible involvement of endoplasmic reticulum stress in inducing apoptosis. Long-term (7–21 days) incubation with BAC (0.2–0.6 μg/mL) did not affect cell viability but led to epithelial-mesenchymal transition (EMT) as shown by the decrease of E-cadherin and the increase of N-cadherin, fibronectin, and vimentin, caused by the upregulation of EMT transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2. Therefore, we conclude that apoptosis could be an important mechanism of acute BAC cytotoxicity in lung epithelial cells, and chronic exposure to BAC even at sub-lethal doses can promote pulmonary EMT.


Introduction
Benzalkonium chloride (BAC), a mixture of n-alkylbenzyldimethylammonium chlorides, is an antimicrobial agent used in agricultural, medical, and household products as a disinfectant,

Cell Viability Assay
Cell viability was examined by MTT assay as described previously [29]. Briefly, the cells in a 96-well plate were treated with different concentrations of BAC and then incubated with 0.5 mg/mL MTT at 37 • C for 1 h. The absorbance of the converted dye was measured at 540 nm using the MULTISKAN GO reader (Thermo Scientific, Sunnyvale, CA, USA), and the results were expressed as a percentage of viable BAC-treated cells normalized to the percentage of untreated cells.

LDH Leakage Assay
Cell cytotoxicity was measured using the DG-LDH500 kit (DoGenBio, Seoul, Korea). The cells were grown to 90% confluency, treated with different concentrations of BAC for 30 min. The 10 µL supernatant was collected and mixed with LDH Reaction Mixture (100 µL). After incubation for 30 min at room temperature (RT), the absorbance was measured at 450 nm using MULTISKAN GO reader (Thermo Scientific).

Annexin V Assay and PI Staining Analysis of Apoptosis
Apoptosis was determined according to the instruction of Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Bedford, MA, USA) as we reported previously [29]. The stained cells by annexin V-FITC and propidium iodide (PI) were analyzed by Becton Dickinson FACSscan flow cytometer (BD Biosciences, San Jose, CA, USA) and BD FACSDiva software (BD Biosciences).

Caspase-3 Activity
The activity of caspase-3 was determined in the same manner as we reported [30]. The fluorescence (Ex380, Em460) by cleavage of substrates Ac-DEVD-AMC was measured using Promega Glomax microplate reader (Promega, Madison, WI, USA).

Examination of Mitochondrial Membrane Potential (MMP, ∆ψ)
The mitochondrial membrane potential (MMP, ∆ψ) was detected by staining of tetraethylbenzimidazolylcarbocyanine iodide (JC-1) according to the protocol we reported [29]. Stained cells were subjected to FACSscan flow cytometer and BD FACSDiva software (BD Biosciences)

Immunofluorescence Staining
Cells were fixed with 4% paraformaldehyde (PFA) for 20 min and permeabilized in 0.2% Triton X-100 for 10 min at RT. Cells were incubated with Alexa-488-conjugated E-cadherin (1:500, Cell Signaling Technology, Danver, MA, USA) and Alexa-555-conjugated vimentin antibodies (1:100, Cell Signaling Technology) at 4 • C overnight and staining was detected by green and red fluorescence, respectively. Nuclei were stained blue using DAPI. The cells were examined under a confocal microscope.

Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Isolation of total RNA using the Direct-zol™ RNA kit (Zymo Research, Orange, CA, USA) and synthesis of cDNA using the iScript™ cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) followed the manufacturer's protocol. The transcripts were amplified using the SensiFAST SYBR qPCR mix (Bioline, London, UK) [31]. Table 1 shows the primers used for amplification.

Cell Migration Assay
We utilized Costar (Cambridge, MA, USA) plates containing polycarbonate filter inserts with a pore size of 8 µm. NCI-H358 cells were placed in the upper chamber containing serum-free medium, while the lower chamber contained medium with 10% FBS. The transwell chambers were incubated for 16 h and the number of cells that moved to the bottom of the insert were fixed, stained with crystal violet, and counted to determine the relative migration.

Statistical Analysis
Results were expressed as mean ± SD and were analyzed by two-tailed Student's t-test (statistical significance at p < 0.05).

BAC-Induced Cytotoxicity in Lung Epithelial Cells
BAC induced concentration-and time-dependent cytotoxicity in H358 cells (Figure 1). At higher concentrations (>10 µg/mL) of BAC, cell viability was dramatically decreased after 30 min incubation ( Figure 1A). The lactate dehydrogenase (LDH) level in the medium, which indicates cell membrane damage, was significantly increased after 30 min exposure to BAC (>4 µg/mL) ( Figure 1B). At 24 h treatment, 1-40 µg/mL of BAC decreased cell viability, and >80% of the cells were dead at >4 µg/mL of BAC ( Figure 1A,C). The IC50 of BAC at 30 min and 24 h incubation periods were 7.1 µg/mL and 1.5 µg/mL, respectively.

Induction of Necrosis and Apoptosis by BAC in Lung Epithelial Cells
Since BAC treatment rapidly damaged the cell membranes ( Figure 1B), we hypothesized that necrotic cell lysis could be the major cell death pathway of cells exposed to BAC at higher concentrations. Cell death processes were monitored by fluorescence-activated cell sorting (FACS) analysis using Annexin V and propidium iodide (PI) after 24 h of BAC incubation. BAC (4-40 μg/mL) increased both necrotic and apoptotic cell death in a dose-dependent manner (Figure 2A-D). However, apoptosis was predominantly induced in cells treated with cytotoxic concentrations (2-40 μg/mL) of BAC (Figure 2A-D). At the highest concentration of BAC (40 μg/mL), most of the cells were dead, and the percentage of apoptotic and necrotic cells were 60.5% and 39.1%, respectively (Figure 2A-D). To determine the effect of BAC on apoptosis in early time period, caspase-3 activity, an indicator of apoptosis, was measured at 6 h and 12 h after BAC treatment. BAC (2-40 μg/mL) increased caspase-3 activity in a dose-and time-dependent manner ( Figure 2E). These results suggest that apoptosis, rather than necrosis, might be the major cell death pathway induced by BAC in lung epithelial cells.

Induction of Necrosis and Apoptosis by BAC in Lung Epithelial Cells
Since BAC treatment rapidly damaged the cell membranes ( Figure 1B), we hypothesized that necrotic cell lysis could be the major cell death pathway of cells exposed to BAC at higher concentrations. Cell death processes were monitored by fluorescence-activated cell sorting (FACS) analysis using Annexin V and propidium iodide (PI) after 24 h of BAC incubation. BAC (4-40 µg/mL) increased both necrotic and apoptotic cell death in a dose-dependent manner (Figure 2A To determine the effect of BAC on apoptosis in early time period, caspase-3 activity, an indicator of apoptosis, was measured at 6 h and 12 h after BAC treatment. BAC (2-40 µg/mL) increased caspase-3 activity in a dose-and time-dependent manner ( Figure 2E). These results suggest that apoptosis, rather than necrosis, might be the major cell death pathway induced by BAC in lung epithelial cells.

Mitochondrial Depolarization and Activation of the Apoptotic Signaling Pathway by BAC
To elucidate the cellular mechanism of apoptosis, mitochondrial membrane potential and apoptotic protein levels in the cells were determined after incubation of cells with BAC for 24 h. Mitochondrial depolarization, an important cause of apoptosis, was observed in 14.9% and 36.2% of cells exposed to 2 and 4 μg/mL of BAC, respectively ( Figure 3A,B). The levels cellular proteins involved in the apoptotic cascade were also significantly altered by BAC treatment. The protein levels of Bcl2-associated X apoptosis regulator (Bax) and its transcriptional activator p53 were elevated, while that of the anti-apoptotic B-cell lymphoma 2 (Bcl2) was reduced by BAC ( Figure 3C). Bcl2 is known to inhibit Bax which forms pores on the mitochondrial outer membrane [18,32]. Thus, the increased Bax and decreased Bcl2 expression levels could lead to decreased mitochondrial membrane potential. The protein levels and activity of cleaved caspase-3, an apoptosis executioner degrading the cellular components, were increased by BAC (2-4 μg/mL) ( Figure 3C,D). The cleaved poly (ADPribose) polymerase (PARP) level, a biomarker of caspase activity [33,34], was also elevated ( Figure  3C). Various stress signals can activate the expression of p53 which then initiates apoptosis via inducing Bax, and arrests cell cycle by elevating p21 expression [18,35]. In the present study, the increased expression levels of p53 and p21 by BAC ( Figure 3C) suggest that BAC treatment induced significant cellular stress resulting in apoptosis in lung cells.

Mitochondrial Depolarization and Activation of the Apoptotic Signaling Pathway by BAC
To elucidate the cellular mechanism of apoptosis, mitochondrial membrane potential and apoptotic protein levels in the cells were determined after incubation of cells with BAC for 24 h. Mitochondrial depolarization, an important cause of apoptosis, was observed in 14.9% and 36.2% of cells exposed to 2 and 4 µg/mL of BAC, respectively ( Figure 3A,B). The levels cellular proteins involved in the apoptotic cascade were also significantly altered by BAC treatment. The protein levels of Bcl2-associated X apoptosis regulator (Bax) and its transcriptional activator p53 were elevated, while that of the anti-apoptotic B-cell lymphoma 2 (Bcl2) was reduced by BAC ( Figure 3C). Bcl2 is known to inhibit Bax which forms pores on the mitochondrial outer membrane [18,32]. Thus, the increased Bax and decreased Bcl2 expression levels could lead to decreased mitochondrial membrane potential. The protein levels and activity of cleaved caspase-3, an apoptosis executioner degrading the cellular components, were increased by BAC (2-4 µg/mL) ( Figure 3C,D). The cleaved poly (ADP-ribose) polymerase (PARP) level, a biomarker of caspase activity [33,34], was also elevated ( Figure 3C). Various stress signals can activate the expression of p53 which then initiates apoptosis via inducing Bax, and arrests cell cycle by elevating p21 expression [18,35]. In the present study, the increased expression levels of p53 and p21 by BAC ( Figure 3C) suggest that BAC treatment induced significant cellular stress resulting in apoptosis in lung cells.

ER Stress Induced by BAC in Lung Epithelial Cells
ER stress is a cellular condition of impaired protein folding function in the ER The accumulation of unfolded/misfolded proteins in the ER activates unfolded protein response (UPR) which improves protein folding, inhibits protein synthesis, and degrades misfolded proteins for maintaining ER homeostasis. However, when the ER function cannot be restored due to severe and prolonged ER stress, apoptosis is triggered to eliminate the stressed cells, thereby protecting the organelle [36,37]. In the present study, the levels of ER stress markers, such as binding immunoglobulin protein (BiP, also called GRP78) and C/EBP homologous protein (CHOP) were increased by BAC (2-4 μg/mL) ( Figure 4). The levels of the UPR sensor protein, inositol requiring enzyme 1α (IRE1α), and the downstream signaling protein, phospho-c-Jun N-terminal kinase (p-JNK), were also enhanced in BAC-treated cells (Figure 4). Since activated JNK is known to upregulate pro-apoptotic genes [38], BAC-induced ER stress could be associated with the induction of apoptosis in lung epithelial cells.

ER Stress Induced by BAC in Lung Epithelial Cells
ER stress is a cellular condition of impaired protein folding function in the ER The accumulation of unfolded/misfolded proteins in the ER activates unfolded protein response (UPR) which improves protein folding, inhibits protein synthesis, and degrades misfolded proteins for maintaining ER homeostasis. However, when the ER function cannot be restored due to severe and prolonged ER stress, apoptosis is triggered to eliminate the stressed cells, thereby protecting the organelle [36,37]. In the present study, the levels of ER stress markers, such as binding immunoglobulin protein (BiP, also called GRP78) and C/EBP homologous protein (CHOP) were increased by BAC (2-4 µg/mL) (Figure 4). The levels of the UPR sensor protein, inositol requiring enzyme 1α (IRE1α), and the downstream signaling protein, phospho-c-Jun N-terminal kinase (p-JNK), were also enhanced in BAC-treated cells (Figure 4). Since activated JNK is known to upregulate pro-apoptotic genes [38], BAC-induced ER stress could be associated with the induction of apoptosis in lung epithelial cells.

EMT Induced by BAC in Lung Epithelial Cells
H358 cells incubated with 0.2-0.6 μg/mL of BAC did not affect the cell viability up to 21 days (data not shown). However, incubation of cells with BAC for 7, 14, and 21 days altered the levels of EMT-associated proteins in the cells. The expression of E-cadherin, which is responsible for cellular adhesion was decreased, but the expression of vimentin, a cytoskeletal component found in mesenchymal cells, was increased by BAC in a concentration-and time-dependent manner ( Figure  5A-C). The decreased E-cadherin mRNA and increased vimentin mRNA might account for the altered expressions of these proteins ( Figure 5D,G). Moreover, the mRNA levels of N-cadherin, a transmembrane glycoprotein that leads to cell invasion, and fibronectin, a component of the ECM, were increased by BAC (Figure 5E,F). In addition, microscopic examination after immunofluorescence staining showed that after 21 days of BAC treatment, the E-cadherin fluorescence intensity (green) in H358 cells was decreased, while that of vimentin (red) was increased in a concentration-dependent manner ( Figure 6). The expression levels of EMT-inducing transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2, were increased by 21 days of BAC exposure in a dose-dependent manner as observed by the elevated expression levels of their mRNA (Figure 7). To confirm the induction of EMT, the migratory properties of H358 cells were examined after incubation of cells with BAC (0.2-0.4 μg/mL) for 21 days using a transwell chamber system. Significantly increased migration of cells through the membrane was observed at 16 h in BAC-treated cells compared to untreated cells (Figure 8). These results clearly show the EMT promoting effect of BAC in lung epithelial cells.

EMT Induced by BAC in Lung Epithelial Cells
H358 cells incubated with 0.2-0.6 µg/mL of BAC did not affect the cell viability up to 21 days (data not shown). However, incubation of cells with BAC for 7, 14, and 21 days altered the levels of EMT-associated proteins in the cells. The expression of E-cadherin, which is responsible for cellular adhesion was decreased, but the expression of vimentin, a cytoskeletal component found in mesenchymal cells, was increased by BAC in a concentration-and time-dependent manner ( Figure 5A-C). The decreased E-cadherin mRNA and increased vimentin mRNA might account for the altered expressions of these proteins ( Figure 5D,G). Moreover, the mRNA levels of N-cadherin, a transmembrane glycoprotein that leads to cell invasion, and fibronectin, a component of the ECM, were increased by BAC ( Figure 5E,F). In addition, microscopic examination after immunofluorescence staining showed that after 21 days of BAC treatment, the E-cadherin fluorescence intensity (green) in H358 cells was decreased, while that of vimentin (red) was increased in a concentration-dependent manner ( Figure 6). The expression levels of EMT-inducing transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2, were increased by 21 days of BAC exposure in a dose-dependent manner as observed by the elevated expression levels of their mRNA (Figure 7). To confirm the induction of EMT, the migratory properties of H358 cells were examined after incubation of cells with BAC (0.2-0.4 µg/mL) for 21 days using a transwell chamber system. Significantly increased migration of cells through the membrane was observed at 16 h in BAC-treated cells compared to untreated cells ( Figure 8). These results clearly show the EMT promoting effect of BAC in lung epithelial cells.

EMT Induced by BAC in Lung Epithelial Cells
H358 cells incubated with 0.2-0.6 μg/mL of BAC did not affect the cell viability up to 21 days (data not shown). However, incubation of cells with BAC for 7, 14, and 21 days altered the levels of EMT-associated proteins in the cells. The expression of E-cadherin, which is responsible for cellular adhesion was decreased, but the expression of vimentin, a cytoskeletal component found in mesenchymal cells, was increased by BAC in a concentration-and time-dependent manner ( Figure  5A-C). The decreased E-cadherin mRNA and increased vimentin mRNA might account for the altered expressions of these proteins ( Figure 5D,G). Moreover, the mRNA levels of N-cadherin, a transmembrane glycoprotein that leads to cell invasion, and fibronectin, a component of the ECM, were increased by BAC ( Figure 5E,F). In addition, microscopic examination after immunofluorescence staining showed that after 21 days of BAC treatment, the E-cadherin fluorescence intensity (green) in H358 cells was decreased, while that of vimentin (red) was increased in a concentration-dependent manner ( Figure 6). The expression levels of EMT-inducing transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2, were increased by 21 days of BAC exposure in a dose-dependent manner as observed by the elevated expression levels of their mRNA (Figure 7). To confirm the induction of EMT, the migratory properties of H358 cells were examined after incubation of cells with BAC (0.2-0.4 μg/mL) for 21 days using a transwell chamber system. Significantly increased migration of cells through the membrane was observed at 16 h in BAC-treated cells compared to untreated cells (Figure 8). These results clearly show the EMT promoting effect of BAC in lung epithelial cells.  is used as a loading control. mRNA levels of (D) E-cadherin, (E) N-cadherin, (F) fibronectin, and (G) vimentin after 21 days of exposure to BAC. Statistical significance was determined by Student's t-test (*p < 0.05, ***p < 0.001, compared to untreated cells).   is used as a loading control. mRNA levels of (D) E-cadherin, (E) N-cadherin, (F) fibronectin, and (G) vimentin after 21 days of exposure to BAC. Statistical significance was determined by Student's t-test (*p < 0.05, ***p < 0.001, compared to untreated cells).

Discussion
The cytotoxicity of BAC has been reported to be due to its chemical structure, composed of NR4 + , a positively charged nitrogen atom and long alkyl groups [2,4]. The hydrophobic carbon chains can easily penetrate the lipid bilayer of the cell membrane, and the charged nitrogen remaining at the membrane surface perturbs the charge distribution. As a result, hydrophilic voids are created, phospholipids are solubilized, and finally the membrane structure is destroyed [2,4]. In alveolar and bronchial epithelial cells, BAC (1-100 μg/mL) treatment has been shown to induce acute cytotoxicity with cell membrane disruptions [14,16]. Inhalation of BAC damages airway epithelium resulting in the release of proteins and LDH to the bronchioalveolar spaces in rodents [11,12,15]. Thus, necrotic cell lysis has been considered to be the primary consequence of BAC-induced pulmonary toxicity. In the present study, BAC treatment rapidly destroyed the cell membranes ( Figure 1B). However, the major cell death pathway in H358 cells exposed to cytotoxic doses of BAC for 24 h was apoptosis rather than necrosis (Figure 2). These results suggest that apoptosis also could be an important mechanism of BAC-induced lung epithelial injury in addition to necrosis.
Apoptosis in injured lung airway has a beneficial role in maintaining organ homeostasis by removing damaged cells. However, excessive apoptosis contributes to the pathogenesis of pulmonary diseases [39]. One recently published paper shows that BAC (20-40 μg/mL) triggers apoptotic pathway via caspase-3 activation and PARP cleavage in human alveolar epithelial (A549) cells after 0.5 h, 1 h, and 4 h incubations [40]. However, other cellular events involved in apoptotic processes were not examined [40]. In the present study, BAC (2-4 μg/mL) appears to promote mitochondria-dependent apoptosis. In normal cells, the anti-apoptotic proteins Bcl2 and B-cell lymphoma-extra large (Bcl-xL) bind to Bax and Bcl2 antagonist/killer 1 (Bak) on the mitochondrial outer membrane. However, under stress conditions, Bax and Bak are separated from Bcl2 or Bcl-xL to form pores allowing the release of mitochondrial cytochrome c. The cytosolic cytochrome c triggers the activation of caspase-9 which then cleaves and activates caspase-3 to degrade cellular components [18,32]. In our results, the decreased Bcl2 and increased Bax expression levels could result in the formation of mitochondrial pores which collapse the membrane potential (ΔΨ), a driving force for ATP synthesis. Cells that are stressed by DNA damage, oxidative stress, and ER stress activate the expression of p53 which then induces the transcription of Bax [18,32,39,41], but inhibits Bcl2

Discussion
The cytotoxicity of BAC has been reported to be due to its chemical structure, composed of NR4 + , a positively charged nitrogen atom and long alkyl groups [2,4]. The hydrophobic carbon chains can easily penetrate the lipid bilayer of the cell membrane, and the charged nitrogen remaining at the membrane surface perturbs the charge distribution. As a result, hydrophilic voids are created, phospholipids are solubilized, and finally the membrane structure is destroyed [2,4]. In alveolar and bronchial epithelial cells, BAC (1-100 µg/mL) treatment has been shown to induce acute cytotoxicity with cell membrane disruptions [14,16]. Inhalation of BAC damages airway epithelium resulting in the release of proteins and LDH to the bronchioalveolar spaces in rodents [11,12,15]. Thus, necrotic cell lysis has been considered to be the primary consequence of BAC-induced pulmonary toxicity. In the present study, BAC treatment rapidly destroyed the cell membranes ( Figure 1B). However, the major cell death pathway in H358 cells exposed to cytotoxic doses of BAC for 24 h was apoptosis rather than necrosis ( Figure 2). These results suggest that apoptosis also could be an important mechanism of BAC-induced lung epithelial injury in addition to necrosis.
Apoptosis in injured lung airway has a beneficial role in maintaining organ homeostasis by removing damaged cells. However, excessive apoptosis contributes to the pathogenesis of pulmonary diseases [39]. One recently published paper shows that BAC (20-40 µg/mL) triggers apoptotic pathway via caspase-3 activation and PARP cleavage in human alveolar epithelial (A549) cells after 0.5 h, 1 h, and 4 h incubations [40]. However, other cellular events involved in apoptotic processes were not examined [40]. In the present study, BAC (2-4 µg/mL) appears to promote mitochondria-dependent apoptosis. In normal cells, the anti-apoptotic proteins Bcl2 and B-cell lymphoma-extra large (Bcl-xL) bind to Bax and Bcl2 antagonist/killer 1 (Bak) on the mitochondrial outer membrane. However, under stress conditions, Bax and Bak are separated from Bcl2 or Bcl-xL to form pores allowing the release of mitochondrial cytochrome c. The cytosolic cytochrome c triggers the activation of caspase-9 which then cleaves and activates caspase-3 to degrade cellular components [18,32]. In our results, the decreased Bcl2 and increased Bax expression levels could result in the formation of mitochondrial pores which collapse the membrane potential (∆Ψ), a driving force for ATP synthesis. Cells that are stressed by DNA damage, oxidative stress, and ER stress activate the expression of p53 which then induces the transcription of Bax [18,32,39,41], but inhibits Bcl2 expression [17]. Moreover, p53 can directly bind to the mitochondrial Bcl2 leading to the release of Bax and Bak [42,43]. In addition, p53 arrests cell cycle via inducing p21 expression thereby allowing cells to recover, but, triggers apoptosis under the severe stress conditions [35,44]. It has been reported that BAC induces DNA damage and oxidative stress in human lung epithelial cells [14,16], suggesting that these cellular stress events promoted by BAC could be possible reasons for the increased expression of p53 ( Figure 3C). PARP catalyzes the transfer reaction of ADP-ribose for DNA repair, and this enzyme can be cleaved and inactivated by caspase-3 [45]. Since PARP-mediated reaction consumes ATP, the inactivation of PARP ( Figure 3C) could preserve ATP for the energy-dependent apoptotic process, as observed in our study. BAC-induced apoptosis via mitochondrial depolarization, cytochrome c release, activation of caspases, and PARP cleavage has been reported in human corneal and conjunctival epithelial cell lines [21,46], suggesting similar mitochondria-involved apoptogenic effect of BAC in mammalian cells.
ER is an organelle in which newly synthesized proteins are folded by ER-resident proteins such as foldases, calreticulin, and chaperones [36,37,47]. BiP, an ER chaperone, binds to hydrophobic residues of proteins to prevent their misfolding [47]. BiP also binds to three UPR sensor proteins, IRE1α, activating transcription factor-6 (ATF6), and PKR-like eukaryotic initiation factor 2a kinase (PERK) to maintain them in an inactive state [36,37]. However, due to ER stress, BiP preferentially associates with unfolded/misfolded proteins, and releases the UPR sensor proteins thereby activating them [48]. Activated IRE1α degrades RNA to attenuate protein synthesis and mediates the translation of X box-binding protein 1 (XBP1) [36,37,47]. XBP1 then activates genes involved in ER biogenesis, ER-associated degradation, and chaperone synthesis such as synthesis of BiP [49,50]. CHOP is induced via the activation of PERK and ATF6, and the latter is also known to promote the expression of BiP [51,52]. Thus, in the present study, the increased levels of IRE1α, BiP, and CHOP proteins clearly indicate BAC-induced ER stress in lung epithelial cells.
Intensive and sustained UPR signaling can eventually trigger apoptosis [36,37,47]. CHOP is a pro-apoptotic transcription factor that inhibits Bcl2 but induces the expression of the apoptotic initiator Bim, resulting in the activation of the caspase-9 and caspase-3 axis [36,37]. Activated IRE1α forms a complex with tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase1 (ASK1), and this complex subsequently phosphorylates JNK [36,37,47]. The activated JNK phosphorylates ER membrane Bcl2 resulting in the release of Bax and Bak to form pores on the ER membrane [53]. Through these pores, ER calcium is released and taken up by mitochondria leading to the depolarization of this organelle [36,37,47]. Since p-JNK is also known to activate p53 [36], the increased levels of IRE1α and p-JNK by BAC (Figure 4) indicate the possibility of ER stress-mediated apoptosis. Moreover, the elevated Bax and reduced Bcl2 protein levels in BAC-treated cells ( Figure 3C) could possibly cause pore formation on the ER membrane. Previously, BAC-exposed zebrafish (Danio rerio) eleuthero-embryos and liver cells showed activated gene expression of ATF6, BiP, CHOP, and XBP1 resulting in apoptosis via the increased expression of Bax, caspase-9 and p53 [54]. Thus, in our study, BAC-promoted ER stress could be directly associated with lung epithelial apoptosis. Nevertheless, the mechanism by which BAC induced ER stress in cells, and whether ER stress was the critical cause of apoptosis remains unclear.
EMT has been observed in wounded and inflamed airway epithelia [55,56]. The potent inducer of EMT is transforming growth factor-β (TGF-β), which is secreted by inflammatory cells into the bronchioalveolar space [55,56]. This cytokine binds to epithelial transmembrane TGF-β receptors that phosphorylate Smad2 or Smad3 to form a complex with Smad4. This complex translocates into the nucleus to express EMT-inducing transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2, which are responsible for the downregulation of E-cadherin, and the upregulation of N-cadherin, fibronectin, vimentin, and collagen [55][56][57]. Cells undergoing EMT break down tight junctions and lose cell-cell adhesion due to loss of E-cadherin expression and gain migratory potential due to increased N-cadherin expression [56,57]. Acquiring motility and a flat shape have beneficial roles in tissue repair, since the cells can migrate to the wounded airway epithelium and spread out for reestablishing the intact barrier function [56,58]. In the present study, although BAC (0.2-0.6 µg/mL) treatment for 7-21 days induced EMT via the upregulation of EMT transcription factors in H358 cells, cell viability was not altered. It has been reported that non-toxic concentrations (0.5-0.75 µg/mL) of BAC elevated TGF-β1, TGF-β receptor1, Smad3, and phospho-Smad3 protein levels in conjunctival fibroblasts [59]. Since lung epithelial cells exposed to toxic stimuli can express TGF-β, which then promotes EMT by its autocrine action [27], the activation of the TGF-β signaling pathway could be a possible reason for BAC-promoted EMT in the present study. Moreover, ER stress by BAC (Figure 4) treatment could result in EMT due to ER stress inducers, such as tunicamycin and thapsigargin, in lung epithelial cells [60,61]. Previously, it has been reported that inhalation of BAC aerosol for 2 weeks caused pulmonary fibrosis in rats [15]. In the present study, long-term incubation of cells with BAC led to increased expression levels of ECM components, fibronectin, and vimentin ( Figures 5 and 6) suggesting the fibrogenic effect of BAC through EMT. Nevertheless, the role of BAC-promoted EMT in animal lung fibrosis is unclear.

Conclusions
In conclusion, we document, that BAC can induce necrosis, apoptosis, ER stress, and EMT in lung epithelial cells in a time-and concentration-dependent manner. These findings can contribute to the understanding of cellular responses in BAC-exposed lung epithelium, but the exact effects of this substance on the animal lung airway remain unclear. Therefore, further in vivo experiments such as rodent inhalation studies are required to clarify the apoptotic and EMT inducing effects of BAC in the pulmonary organs of animals.